Citation or identification of any reference in section 2 or any section of this application shall not be construed as an admission that such reference is available as prior art to the present invention.
A combinatorial library is a collection of multiple species of chemical compounds comprised of smaller subunits or monomers. Combinatorial libraries come in a variety of sizes, ranging from a few hundred to several thousand species of chemical compounds. There are also a variety of library types, including oligomeric and polymeric libraries comprised of compounds such as peptides, carbohydrates, oligonucleotides, and small organic molecules, etc. Such libraries have a variety of uses, such as identifying and organic molecules, etc. Such libraries have a variety of uses, such as identifying and characterizing ligands capable of binding an acceptor molecule or mediating a biological activity of interest.
The library compounds may comprise any type of molecule of any type of subunits or monomers, including polymers wherein the monomers are chemically connected by any sort of chemical bond such as covalent, ionic, coordination, chelation bonding, etc., which those skilled in the art will recognize can be synthesized on a solid-phase support. The term polymer as used herein includes those compounds conventionally called heteropolymers, i.e., arbitrarily large molecules composed of varying monomers, wherein the monomers are linked by means of a repeating chemical bond or structure. The polymers of the invention of this types are composed of subunits or monomers that can include any bi-functional organic or herteronuclear molecule including, but not limited to amino acids, amino hydroxyls, amino isocyanates, diamines, hydroxycarboxylic acids, oxycarbonylcarboxylic acids, aminoaldehydes, nitroamines, thioalkyls, and haloalkyls. In the disclosure of the present invention, the terms “monomer,” “subunits” and “building blocks” will be used interchangeably to mean any type of chemical building block of molecule that may be formed upon a solid-phase support.
Various techniques for synthesizing libraries of compounds on solid-phase supports are known in the art. Solid-phase supports are typically polymeric objects with surfaces that are functionalized to bind with subunits or monomers to form the compounds of the library. Synthesis of one library typically involves a large number of solid-phase supports. Solid-phase supports known in the art include, among others, polystyrene resin beads, cotton threads, and membrane sheets of polytetrafluoroethylene (“PTFE”).
To make a combinatorial library, solid-phase supports are reacted with a one or more subunits of the compounds and with one or more numbers of reagents in a carefully controlled, predetermined sequence of chemical reactions. In other words, the library subunits are “grown” on the solid-phase supports. The larger the library, the greater the number of reactions required, complicating the task of keeping track of the chemical composition of the multiple species of compounds that make up the library. Thus, it is important to have methods and apparatuses which facilitate the efficient production of large numbers of chemical compounds, yet allow convenient tracking of the compounds over a number of reaction steps necessary to make the compounds.
One method of making combinatorial libraries is described in U.S. Pat. No. 5,510,240 to Lam et al. (“Lam '240 patent”), the disclosure of which is incorporated herein by reference in its entirety. More specifically, the Lam '240 patent discloses a split and mix method of synthesizing combinatorial libraries of bio-oligomers on resin beads, in certain embodiments of which the library contains all possible combinations of monomer subunits of which the bio-oligomers are composed. Although there may be several resin beads containing the same species of bio-oligomer, each resin bead contains only one species of bio-oligomer.
Another example of a method of making combinatorial libraries on divisible solid-phase supports is described in U.S. Pat. No. 5,688,696 to Lebl (“Lebl '696 patent”), the disclosure of which is incorporated herein by reference in its entirety. In the method disclosed in the Lebl '696, each of a set of predetermined species of test compounds is present on a predetermined number of solid-phase supports—preferably on only one—and each solid-phase support has only a single species of test compound.
The use of radio-frequency identification (“RFID”) chips to record the steps of library synthesis is also known. See, for example, U.S. Pat. Nos. 5,741,462, 5,770,455, and 5,751,629, as well as WO 98/15826.
A method and apparatus for synthesis of a combinatorial library using a 3-D array of reaction zones is provided in Glaxo's WO 99/32219 (“Glaxo Application”). This application discloses stackable frames having a plurality of holes. Membranes, which act as the solid supports, are trapped between stacked frames, and these membranes are exposed at the frame holes. In an alternative embodiment, solid support beads are placed on flow-through sieves that allow flow-through of reagents around the support beads. Reagents are pumped in from the top and vacated at the bottom or, alternatively, pumped in from the bottom and vacated at the top. The apparatus disclosed allows reagents to be delivered to groups of supports in the X-Z planes or in the Y-Z planes during synthesis steps.
The Glaxo Application also employs a 3-D (X-Y-Z) array of supports. However, instead of using a containment apparatus having true wells in which solid supports are stacked, the Glaxo method employs stackable 2-D (X-Y) frames. The Glaxo Application discloses two distinct embodiments of stackable frame structures. One embodiment sandwiches a membrane between stacked frames, the frames having a plurality of holes. The membranes are solid-phase supports which are held between the frames. The frame holes expose the membranes. The membranes also have holes to allow reagents to pass through the layers of membranes and contact other membranes in the vertical “column” of the array. Another embodiment has sieves in place of the membranes, and free solid supports are placed on each sieve between the frames. The sieves allow reagents to flow vertically from top to the bottom of the stacked 3-D array contacting a vertical column of solid-phase supports resting on sieves.
A major disadvantage with Glaxo's apparatus and method, however, is that after the synthesis is completed, the solid supports, whether as the membrane or the solid-phase support beads suspended on the sieve, are not easily freed from the stacked array while retaining their spatial identities. The frames must be taken apart one by one to gain access to the supports and to provide some means to retain the identities of each support. This requires a burdensome additional step that makes the apparatuses disclosed less attractive for commercial production of libraries.
While methods exist in the art that can be used to produce a library of compounds, there is still a need for methods and apparatuses effective for commercial use to build a large library of compounds quickly and with a minimum of cost. Thus, there is still a need for alternative methods of synthesis that use 2-D or 3-D arrays of solid-phase support as part of the synthesis process for the purpose of commercially making large libraries of compounds efficiently.
Moreover, there is still a need for apparatuses and methods for efficiently synthesizing extremely large libraries, e.g., greater than 100,000 compounds, using 2-D or 3-D arrays as tools in the synthesis.